To investigate the role of dietary folic acid in dideazatetrahydrofolate (DDATHF) sensitivity, reduced folates were estimated in plasma and tissue of mice following dietary depletion and repletion. Previous studies showed that DDATHF, a new folate antagonist targeted against glycinamide ribonucleotide transformylase, produced unexpectedly severe toxicity in humans compared with mice. However, toxicity in the animal model also became pronounced upon the removal of folic acid from the diet. Further, modest dietary restoration of folic acid in the drinking water showed that toxicity could be alleviated while antitumor activity was maintained. To investigate the role of dietary folic acid levels on tissue folates in this system, all the natural reduced folates were evaluated by a ternary complex based assay in mice placed on folic acid deplete and replete diets. After 2 weeks on a folic acid deplete diet, total plasma folate had decreased by 85%, whereas red blood cell, liver, and intestinal folate fell by only 50%. Repletion of folic acid in the drinking water at a low level (0.0003%) caused partial restoration of reduced folates, while a higher repletion level (0.003%) resulted in restoration to control levels or above. Administration of folic acid and leucovorin by oral gavage to DDATHF-treated mice resulted in elevation of tissue folates in mice maintained on folic acid deplete and replete diets. Relatively high levels of folic acid were present in plasma following oral gavage of folic acid, while essentially no [S]5-formyltetrahydrofolate was observed after leucovorin. Reduced folate pools in a subcutaneously implanted mouse mammary adenocarcinoma responded more extensively to dietary folic acid depletion than folate pools in liver. Likewise, these pools were more sensitive to restoration by folic acid or leucovorin. This greater reduced folate response of tumor versus normal tissue, if confirmed in other systems, suggests a possible basis for selective antitumor activity.